Coregulation of Dimorphism and Symbiosis by Cyclic AMP Signaling in the Lichenized Fungus Umbilicaria Muhlenbergii
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Coregulation of dimorphism and symbiosis by cyclic AMP signaling in the lichenized fungus Umbilicaria muhlenbergii Yanyan Wanga,b, Xinli Weia, Zhuyun Bianb, Jiangchun Weia, and Jin-Rong Xub,1 aState Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, China; and bDepartment of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 Edited by Nicholas J. Talbot, The Sainsbury Laboratory, Norwich, United Kingdom, and accepted by Editorial Board Member Sheng Yang He August 10, 2020 (received for review March 18, 2020) Umbilicaria muhlenbergii is the only known dimorphic lichenized (8). In fact, ∼46% of ascomycetes are lichen mycobionts (9), and fungus that grows in the hyphal form in lichen thalli but as yeast a large majority of them belong to class Lecanoromycetes (8). cells in axenic cultures. However, the regulation of yeast-to-hypha For most lichenized fungi, their free-living forms are rarely ob- transition and its relationship to the establishment of symbiosis served in nature (10), although they may be cultured axenically are not clear. In this study, we show that nutrient limitation and under laboratory conditions. In general, the isolated mycobionts hyperosmotic stress trigger the dimorphic change in U. muhlen- grow very slowly in culture and may require weeks of incubation bergii. Contact with algal cells of its photobiont Trebouxia jamesii to form sizable colonies, which are typically compact and pig- induced pseudohyphal growth. Treatments with the cAMP diphos- mented (11). Among all lichenized ascomycetes (>280 genera) phoesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) induced that have been isolated, only a few species are known to produce pseudohyphal/hyphal growth and resulted in the differentiation conidia and ascospores in culture (12–14). of heavily melanized, lichen cortex-like structures in culture, indi- Resynthesis of a lichen thallus is a complex process that begins cating the role of cAMP signaling in regulating dimorphism. To with the physical contact between the mycobiont and photobiont confirm this observation, we identified and characterized two cells. The two symbiotic partners then grow together to form an Gα subunits UmGPA2 and UmGPA3. Whereas deletion of UmGPA2 undifferentiated mass, which further develops into a stratified MICROBIOLOGY had only a minor effect on pseudohyphal growth, the ΔUmgpa3 thallus after a transitional stage (6, 15). The formation of a mature mutant was defective in yeast-to-pseudohypha transition induced lichen thallus is difficult under laboratory conditions, likely due to by hyperosmotic stress or T. jamesii cells. IBMX treatment sup- the requirement of unknown specific environmental or biological pressed the defect of ΔUmgpa3 in pseudohyphal growth. Trans- factors in nature. However, the initial interaction between the G45V Q208L formants expressing the UmGPA3 or UmGPA3 dominant mycobiont and photobiont and structural development have been active allele were enhanced in the yeast-to-pseudohypha transition reported in several species (16–19). Before the physical contact and developed pseudohyphae under conditions noninducible to the stage, the mycobiont exhibits selectivity and compatibility toward wild type. Interestingly, T. jamesii cells in close contact with pseu- – G45V Q208L different phototrophs (20 22). At the interaction stage, fungal dohyphae of UmGPA3 and UmGPA3 transformants often hyphae often increase the production of short lateral branches to collapsed and died after coincubation for over 72 h, indicating that envelop compatible algal cells (23). Some mycobionts, such as improperly regulated pseudohyphal growth due to dominant active Cladonia, Lecanora,andXanthoria, form haustoria or haustorium-like mutations may disrupt the initial establishment of symbiotic inter- action between the photobiont and mycobiont. Taken together, Significance these results show that the cAMP-PKA pathway plays a critical role in regulating dimorphism and symbiosis in U. muhlenbergii. Umbilicaria muhlenbergii istheonlylichenizedspeciesknowntobe cAMP signaling | lichen-forming fungi | dimorphic transition | dimorphic in culture. This study showed that yeast-to-pseudohypha yeast-to-hypha transition | fungal–algal association transition in U. muhlenbergii is associated with symbiosis and reg- ulated by the cAMP-PKA (protein kinase A) pathway. Treatments with a cAMP diphosphoesterase inhibitor induced pseudohyphal/ ichens are symbiotic associations between a fungus (myco- hyphal growth and differentiation of lichen cortex-like tissues. Two Lbiont) and a photosynthetic partner (photobiont), usually Gα subunits were functionally characterized to show the regulation green algae or cyanobacteria. Apart from the two major symbi- of dimorphism by UmGPA3. This is a report on identifying genes onts, other organisms, including bacteria, fungi, and algae, may be important for development and symbiosis in lichenized fungi. This is present and have functions in lichens as a stable symbiotic com- also a report of generating targeted gene deletion mutants in U. munity (1–3). Lichens can thrive in harsh environments and play ∼ muhlenbergii and Lecanoromycetes in general. Because of its rela- important roles in the ecosystem by covering 8% of the terres- tively fast growth rate and amenability to molecular manipulations, trial surface (4, 5). Based on the morphology of their thalli, lichens U. muhlenbergii is uniquely suited for studying fungal–algal inter- can be categorized into different growth types, such as fruticose, actions and initial symbiotic interactions. foliose, and crustose lichens. Typically, lichens have a cortex or outer layer that consists of highly differentiated, densely aggre- Author contributions: J.W. and J.-R.X. designed research; Y.W., X.W., and Z.B. performed gated fungal hyphae or symbiotic complex in which individual research; Y.W., X.W., and Z.B. analyzed data; and Y.W., J.W., and J.-R.X. wrote the paper. hyphae are no longer distinguishable (6, 7). Beneath the upper The authors declare no competing interest. cortex is a layer of photobiont cells that may be arranged in dis- This article is a PNAS Direct Submission. N.J.T. is a guest editor invited by the order or regularly with interwoven hyphae. For most lichens, the Editorial Board. lower surface or cortex may form special structures for tight ad- Published under the PNAS license. herence to the substrate. 1To whom correspondence may be addressed. Email: [email protected]. Although lichenization occurs in species belonging to poly- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ phyletic groups in different phyla, indicating multiple origins of doi:10.1073/pnas.2005109117/-/DCSupplemental. lichenized fungi, the majority (99%) of them are ascomycetes www.pnas.org/cgi/doi/10.1073/pnas.2005109117 PNAS Latest Articles | 1of12 Downloaded by guest on September 27, 2021 structures within photobiont cells (21, 24). However, due to their of the top layers, U. muhlenbergii strain JL3 was isolated from slow growth rate, lichenized fungi are not ready amenable to the medullary layer of lichen thalli collected at Tulaopoding molecular genetic studies. To date, no genes responsible for the Mountain, China. As previously reported (25), strain JL3 grew by establishment of symbiosis have been functionally characterized budding as yeast cells (3.0 to 4.5 × 5.0 to 6.5 μm) in potato at the molecular level. dextrose agar cultures (SI Appendix, Fig. S2). After incubation at Unlike all other cultivated lichen-forming ascomycetes that 25 °C for 14 d, strain JL3 formed whitish colonies with a smooth grow only as hyphae, the isolated mycobiont of the lichen, margin. BLASTn searches and phylogenetic analysis with the Umbilicaria muhlenbergii, grows by budding as yeast cells in axenic rRNA (ribosomal RNA)-ITS (internal transcribed spacer) se- cultures (25). U. muhlenbergii is a foliose lichen that usually grows quence (ITS1 + 5.8S rRNA + ITS2) amplified with primers ITS4 on bare stones at high altitudes. Its photobiont partner is mainly and ITS5 (33) confirmed that it is a U. muhlenbergii isolate (SI Trebouxia jamesii, a green algal species (26). The unicellular yeast Appendix,Fig.S3). The rRNA-ITS sequence of U. muhlenbergii cells of U. muhlenbergii mycobiont grow by budding and form strain JL3 is identical to that of the published strain (25, 34). typical yeast-like colonies (25). Although U. muhlenbergii lives in We also isolated the green algal photobiont T. jamesii from the the yeast form in culture, it exists in the hyphal or pseudohyphal same specimens and verified its identity by sequencing the form in the lichen thallus, indicating that this lichenized fungus rRNA-ITS region (SI Appendix, Fig. S3). Algal cells of T. jamesii must undergo the yeast-to-hypha transition during the establish- are spherical and 10 to 15 μm in diameter. Another green algal ment of symbiosis. U. muhlenbergii, like many other species in the species with a different colony morphology and bigger algal cells genus Umbilicaria, can grow in extreme environmental conditions than T. jamesii was repeatedly isolated from the same lichen and often produces heavily melanized thalli. Ascospores formed in thallus (SI Appendix, Fig. S2), which was not observed in the apothecia (sexual fruiting bodies) are its preferred propagules for earlier report (27). Analysis of the rRNA-ITS sequences showed dispersal instead of the vegetative reproducing mixtures of that